1. Field of the Invention
The present invention relates generally to the field of clinical microbiology. Specifically, the present invention relates to the detection of viable organisms of the Mycobacterium tuberculosis complex using a reverse transcriptase strand displacement amplification assay.
2. Description of the Related Art
The resurgence of tuberculosis in the United States over the past decade and its continued worldwide dominance as a cause of morbidity and mortality (Raviglione et al, 1995) have focused attention on the need for more rapid and reliable means of diagnosis. Traditionally, diagnosis is dependent upon acid-fast staining and culture of the causative agent, Mycobacterium tuberculosis (M. tuberculosis), in broth or on solid media. However, this process may require up to 6 weeks owing to the slow growth rate of the organism. In contrast, nucleic acid amplification assays have the potential to reduce the time for definitive diagnosis to as little as one day. Several assays have been described for the detection of nucleic acid sequences that are specific for the M. tuberculosis complex which comprises M. tuberculosis, M. bovis, M. bovis bacille Calmette-Guerin (BCG), M. africanum and M. microti (Eisenach et al, 1991; Iovannisci et al, 1993; Jonas et al, 1993; Shah et al, 1995; van der Vliet et al, 1993; Walker et al, 1992). Although beneficial to the initial diagnosis of infection, such assays have so far proven unsuitable for monitoring the response of patients to therapy.
Typically, successful treatment of a patient with tuberculosis results in conversion of smears and cultures to negative within 3-4 months. However, recently it has been demonstrated that DNA-based amplification assays such as the Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR) and Strand Displacement Amplification (SDA) are an inappropriate substitute for conventional microbiological methods of patient follow-up since M. tuberculosis DNA may persist for long periods after smears and cultures have become negative (Hellyer et al, 1996). Similarly, a poor correlation has been observed between smear and culture results and those obtained with the Gen-Probe Amplified Mycobacterium Tuberculosis direct Test for M. tuberculosis 16S ribosomal RNA (Moore et al, 996).
In prokaryotic cells, messenger RNA (mRNA) is degraded rapidly with a typical half-life of 3 minutes (Belasco et al, 1986; von Gabain et al, 1983). Consequently an mRNA-based amplification assay is likely to detect only living organisms and thus be a good indicator of therapeutic efficacy. Thus, the prior art is deficient in methods for diagnosis of and determination of efficacy of treatment for M. tuberculosis.